Dose finding study for on-demand HIV pre-exposure prophylaxis for insertive sex in sub-Saharan Africa: results from the CHAPS open label randomised controlled trial

Summary Background The efficacy of on-demand HIV pre-exposure prophylaxis (PrEP) for men in sub-Saharan Africa has not been evaluated, and the on-demand PrEP dosing requirement for insertive sex remains unknown. Methods HIV-negative males 13–24 years, requesting voluntary medical male circumcision (VMMC), were enrolled into an open-label randomised controlled trial (NCT03986970), and randomised 1:1:1:1:1:1:1:1:1 to control arm or one of eight arms receiving emtricitabine-tenofovir disoproxil fumarate (F/TDF) or emtricitabine-tenofovir alafenamide (F/TAF) over one or two days, and circumcised 5 or 21 h thereafter. The primary outcome was foreskin p24 concentrations following ex vivo HIV-1BaL challenge. Secondary outcomes included peripheral blood mononuclear cell (PBMC) p24 concentration, and drug concentrations in foreskin tissue, PBMCs, plasma and foreskin CD4+/CD4-cells. In the control arm, post-exposure prophylaxis (PEP) activity of non-formulated tenofovir-emtricitabine (TFV-FTC) or TAF-FTC was assessed with ex vivo dosing 1, 24, 48 or 72 h post-HIV-1 challenge. Findings 144 participants were analysed. PrEP with F/TDF or F/TAF prevented ex vivo infection of foreskins and PBMCs both 5 and 21 h after PrEP dosing. There was no difference between F/TDF and F/TAF (p24day15 geometric mean ratio 1.06, 95% confidence interval: 0.65–1.74). Additional ex vivo dosing did not further increase inhibition. In the control arm, PEP ex vivo dosing was effective up to 48 post-exposure diminishing thereafter, with TAF-FTC showing prolonged protection compared to TFV-FTC. Participants receiving F/TAF had higher TFV-DP concentrations in foreskin tissue and PBMCs compared with F/TDF, irrespective of dose and sampling interval; but F/TAF did not confer preferential TFV-DP distribution into foreskin HIV target cells. FTC-TP concentrations with both drug regimens were equivalent and ∼1 log higher than TFV-DP in foreskin. Interpretation A double dose of either F/TDF or F/TAF given once either 5 or 21 h before ex vivo HIV-challenge provided protection across foreskin tissue. Further clinical evaluation of pre-coital PrEP for insertive sex is warranted. Funding EDCTP2, 10.13039/100005564Gilead Sciences, 10.13039/501100004359Vetenskapsrådet.


Introduction
Despite cheap and effective HIV testing and prevention, approximately 320,000 men were newly infected in sub-Saharan Africa (SSA) in 2021. 1 HIV prevention efforts in SSA are primarily female-focused, leaving a gap for men gap in HIV services. 2 Men fare worse in HIV testing uptake, ART initiation, 3 engagement and retention in HIV prevention/treatment programmes. [4][5][6] Oral HIV PrEP has achieved tremendous impact in reducing HIV incidence in Europe and Americas but less in Africa, where PrEP availability is variable. 7 Daily and on-demand PrEP are highly effective and recommended by WHO for use in men. 8 Clinical efficacy of on-demand PrEP has only been evaluated in men who have sex with men (MSM), 9 with no data in other populations including young men in SSA. Hence, ondemand PrEP is not included in SSA guidelines, despite men from the region preferring on-demand PrEP, specifically pre-coital PrEP regimes over daily tablets. 10,11 Regimen simplification for on-demand PrEP is desirable to increase adherence and could be appropriate for insertive sex, which is associated with a 10-fold lower risk of HIV acquisition compared to receptive sex. 12 On-demand PrEP has advantages over daily PrEP:

Research in context
Evidence before this study We searched PubMed and Clinicaltrials.gov for combinations of the terms "tenofovir disoproxil fumarate and emtricitabine", "tenofovir alafenamide and emtricitabine", "foreskin", "male genital tract", "peripheral blood mononuclear cells", "HIV preexposure prophylaxis", and "HIV post-exposure prophylaxis", with no restrictions on language or publication date. Seventyone clinical trials on F/TDF or F/TAF have been completed or are ongoing, but none has tested efficacy against HIV-1 infection in foreskin tissue of on-demand PrEP in men in sub-Saharan Africa (SSA). Drug concentrations in human male genital tract have only been evaluated in secretions which do not precisely reflect tissue drug concentrations. No clinical trial comparing the efficacy and time interval from HIV-1 exposure to PEP initiation for F/TDF and F/TAF was identified.

Added value of this study
This clinical trial uses ex vivo challenge methods evaluating dosing requirements for on-demand PrEP in insertive sex using F/TDF and F/TAF. We show that a double dose of either F/TDF or F/TAF PrEP given 5 or 21 h before exposure, is sufficient to prevent HIV acquisition across both foreskin tissue and PBMCs. There was no difference in protection from HIV-1 between F/TDF and F/TAF, however, F/TAF produced higher intracellular drug concentrations in both PBMCs and target cells within foreskin tissue. Both drugs showed significant PEP activity in foreskin explants however, protection was limited after 48 h, with TAF-FTC providing more durable protection compared to TFV-FTC.
Implications of all the available evidence Understanding dosing requirements for insertive sex is necessary to provide guidance for optimal use. The high concentration of protection found in this study of foreskins from men in Uganda and South Africa provides for the data for insertive sex in men from SSA. Our data suggest that ondemand PrEP could be simplified to a double dose of F/TDF or F/TAF given 5-21 h prior to intercourse. The limited efficacy of TFV-FTC or TAF-FTC as PEP when initiated 48 h after HIV exposure supports evidence from rectal tissue that PEP initiation should be within 48 h of potential exposure to HIV. Articles lower number of tablets required; lower risk of drug resistance selection; and fewer adverse events. F/TAF is licenced for daily PrEP but has not been evaluated for on-demand PrEP. TAF is known to achieve up to 4.1-fold higher concentrations of the intracellular active phosphorylated moiety (tenofovir diphosphate; TFV-DP) in PBMCs, 13 and has a more favourable renal and bone toxicity profile than TDF. 14 Understanding how rapidly HIV protection occurs following PrEP dosing is vital for both daily and on-demand PrEP. Whilst the IPERGAY study provides insight for MSM in Europe initiating F/TDF, 9 in other settings and for those initiating F/TAF, data for insertive sex are not known. PEP remains a useful additional strategy to prevent HIV acquisition. 12 However, there is no clinical data guiding how soon after sex PEP must be started for insertive sex and no comparison between TAF and TDF.
Ex vivo challenge models provide a platform to define markers of biological efficacy and have been used to prioritize HIV-1 prevention candidates for Phase 3 trials. 15 Voluntary medical male circumcision (VMMC) has been scaled-up in many high HIV-prevalence settings in SSA as part of prevention efforts. Many young men choose to become circumcised in Africa each year providing a potential research opportunity to utilise foreskins which otherwise would be discarded. We conducted a clinical trial comparing efficacy of F/TDF versus F/TAF dosing prior to VMMC on HIV-infection of foreskin using an ex vivo challenge model. We used foreskin tissue obtained to ascertain dosing schedules, duration of protection against ex vivo challenge, and additional protection afforded by treatment after ex vivo exposure to HIV. This trial simulated the relative protection that could be attained for insertive sex by preand post-exposure regimens.

Study design and participants
HIV negative males aged 13-24 years were recruited from VMMC clinics at Chris Hani Baragwanath Academic Hospital, Soweto, South Africa or Entebbe General Hospital, Entebbe, Uganda. Eligibility criteria included being clinically eligible for VMMC, weighing >35 kg, and being able to give written informed consent. Full eligibility criteria are described in the protocol (appendix: study protocol). This was a randomised controlled trial with nine trial arms: one control arm and eight treated arms that received PrEP prior to VMMC ( Supplementary Fig. S1). 16

Randomization and masking
Participants were randomised in a 1:1:1:1:1:1:1:1:1 ratio to control arm or one of the eight treated arms. Random allocation sequence was generated by an independent statistician using Stata, stratified by country and using block size 9. Participants who were not circumcised following randomisation were excluded according to protocol, and an additional set of randomisation codes generated using the same approach, to ensure a target sample size of 16 evaluable participants per trial arm was attained. Sequentially numbered opaque envelopes labelled with unique randomisation identifier and containing the allocated intervention arm were prepared by two administrators otherwise uninvolved in the study. At time of randomisation, clinical staff opened the sequential envelope and scheduled the participant to receive PrEP (if applicable) and VMMC, as per randomisation arm. Participants and care providers were not blinded to trial arm. Laboratory outcome assessors were blinded until all measurements were completed.

Ethical approvals
Written informed consent was obtained from all participants aged ≥18 years and emancipated minors (in Uganda); for those <18 years and not emancipated minors, assent with parental consent was obtained.

Outcomes
The primary outcome was HIV-1 p24 concentration in participants' foreskin tissue up to day 15 following ex vivo HIV-challenge and evaluated as p24 concentration at day 15 (p24 d15 ), area-under-the-curve of p24 concentrations between days 3-15 (p24 AUC ) following challenge, and slope of the curve fitted to p24 concentrations between days 3-15 (p24 slope ). Reduction of p24 d15 , p24 AUC and negative p24 slope were indicative of protection, i.e. lack of productive HIV-1 infection.
The secondary outcomes included p24 concentrations in PBMCs; drug concentration in foreskin tissue, foreskin CD4+/CD4-cells, blood (plasma and PBMCs); additional effect of ex vivo dosing with the same oral PrEP drug 20 h post-challenge; and ex vivo PEP efficacy in foreskin from participants in the control arm.

Sampling
Study samples were collected during VMMC visit 5 h or 21 h ± 40 min after the last PrEP dosing. Blood and foreskin tissue, including inner and outer, were collected. VMMC was performed using the dorsal slit method according to local guidelines. Tissue was placed immediately in cold culture media DMEM. Samples were immediately transported to the laboratory on ice (median transit time 30 min). Processing of samples was performed immediately upon arrival to the laboratory.

Ex vivo challenge of foreskin tissue and PBMCs
Foreskin was cut into 2 mm 2 explants 17 and cultured in DMEM supplemented with 10% FBS, 2 mM Lglutamine, 2.5 μg of amphotericin B/mL, 100 U of penicillin/mL, 100 μg of streptomycin/mL, and 80 μg of gentamicin/mL (Sigma, St. Louis, MO, USA). Explants were immediately mock treated or challenged with HIV-1 BaL at a high titre (HVT) (10 4 TCID 50 /mL [median tissue culture infective dose/mL]) generally used for ex vivo challenge or at a more biologically relevant low titre (LVT) (2 × 10 2 TCID 50 /mL). After 20 h, explants from treated arms were further dosed ex vivo for 2 h, or not for controls cases, with nonformulated TFV-FTC or TAF-FTC (Gilead, Foster City, CA, USA) matching the drug used for oral dosing, to mimic PrEP regimes including dosing after sexual intercourse, and with the concentrations previously determined in foreskin explants. 18 Ex vivo dosing of explants was performed in a non-polarized manner as described previously 18 at concentrations previously defined in our laboratory. 17,18 Following drug exposure, tissue explants were washed with PBS, and cultured in complete media in the absence of drug.
Explants from the control arm were used as baseline of infection. Additional explants from the control arm were dosed ex vivo with TFV-FTC or TAF-FTC for 2 h at different times post-viral exposure: 1, 24, 48 or 72 h to assess the PEP potency of these drugs. Isolated PBMCs were challenged or not with HIV-1 BaL at a HVT (10 3 TCID 50 /mL) or a LVT (2 × 10 2 TCID 50 /mL), and after 20 h, dosed or not ex vivo with the same drug used for oral dosing. PBMCs from the control arm were not further dosed ex vivo. All PBMCs were then activated with PHA 5 μg/mL and IL-2 20U/mL for 48 h. Foreskin explants and PBMCs were cultured for 15 days with twothirds of culture supernatant harvested at days 3, 7, 11 and 15, and cultures refed with fresh medium. The extent of virus replication was determined by measuring p24 concentration in supernatants at each harvest time point following manufacturer's instructions (Innotest HIV antigen mAb ELISA, catalogue number: 80563, Fujirebio Europe, Belgium). Non-cumulative p24 values were extrapolated using a sigmoidal dose-response (Prism, GraphPad) and reported as p24 d15 and p24 AUC . The p24 slope was calculated during the 15 days of culture. The lower limit of quantification (LLQ) for the assay was 0.02998. p24 concentrations that were below the LLQ of each assay were expressed as half-LLQ values.

Bioanalytical methods
Concentrations (denoted as [drug or metabolite]) of TFV, FTC, and the pro-drug TAF, were measured in plasma and foreskin tissue. Concentrations of the active phosphorylated intracellular metabolitestenofovirdiphosphate (TFV-DP) and emtricitabine-triphosphate (FTC-TP) were determined in PBMCs, foreskin tissue, and isolated foreskin CD4+ and CD4-cells. Analyte quantification was performed using a SCIEX 4500 or 5500 triple quadrupole mass spectrometer (AB Sciex UK Limited; Warrington, UK). Data acquisition and processing were performed using FDA CFR Part 11 compliant SCIEX Analyst and Multiquant software platforms. LC-MS assays were validated in accordance with FDA Bioanalytical Method Validation Guidelines. 19 Relevant bioanalytical method instrumentation and assay validation parameters are summarised in Supplementary Tables S1 and S2 Drug and metabolite concentrations in foreskin were quantified using a ng/sample or pmol/ sample calibration curve and values normalised to ng/g or pmol/g of tissue. Intracellular concentrations of phosphorylated metabolites in PBMCs and CD4± cells were expressed as mol/10 6 cells. Concentrations below Articles the assay LLQ were expressed as half-LLQ values. Concentrations below the assay lower limit of detection (with no visible chromatographic signal) were excluded.

Statistical analysis
The planned sample size of 144 participants was based on feasibility of conducting this number of experiments based on previous ex vivo challenge models. Analysis was done on a per protocol basis. Participant characteristics were summarised by trial arm, using frequency and proportion for categorical variables and mean and standard deviation for continuous variables. All PD and PK outcomes were positively skewed and therefore log-transformed for analysis; logtransformed variables were approximately normally distributed. Outcomes were summarised using geometric means and 95% confidence interval (CI). The relative effect of trial interventions on primary and secondary outcomes was assessed through the following a-priori defined comparisons: (i) any PrEP versus control arm, (ii) FTC-TAF versus FTC-TDF, (iii) 2 + 1 tablets versus 2 tablets, (iv) 21 h between PrEP and VMMC versus 5 h. Further comparisons also assessed the effect of dosage separately for each drug, and the effect of interval, separately for each drug and dosage. Linear regression was used to determine the mean difference and 95% CI for each comparison, with parameters back-transformed to geometric mean ratios (GMR), and p-values determined by likelihood ratio tests. We assessed assumptions underlying linear regression; homoscedasticity was assessed through plotting fitted values against residuals and normality of residuals was assessed through QQ plots. PBMC metabolite concentrations from this study were related to previously defined protective thresholds of greater than 16 fmol/10 6 cells (TFV-DP) and 3.7 pmol/ 10 6 cells (FTC-TP) which were associated with a 90% reduction in risk of HIV-1 acquisition. 20 Additional effect of ex vivo dosing on p24 concentrations in tissue and PBMCs was assessed using paired t-tests, comparing p24 in tissue/PBMCs that received ex vivo dosing with p24 in tissue/PBMC that did not receive ex vivo dosing, with the comparison made within each participant. Ex vivo efficacy of PEP was compared between trial arms using independent t-tests. As a posthoc analysis, we calculated the percentage reduction in p24 at day 15 in both tissue and PBMCs among participants receiving PrEP compared to participants in the control arm.

Role of the funding source
Funders had no role in the data collection, data analysis or data interpretation. In addition, EDCTP2 and Vetenskapsrådet had no role in study design or writing of the report. All authors had full access to all study data and had final responsibility for the decision to submit for publication.

Results
Between 19th October 2019 and 5th March 2021, 178 young men were screened, 17 did not meet eligibility criteria and five did not return for the randomisation visit, leaving 156 randomised participants. Of these, 11 were not circumcised and foreskin from one circumcised participant was given the wrong ex vivo drug in error; these all were judged non-evaluable and excluded from analyses. A total of 144 participants, 16 per trial arm, were analysed (Fig. 1). Characteristics of included participants were similar between trial arms (Table 1). Twenty-one adverse events were reported among 19 participants (Supplementary Table S3) with no serious adverse events.
Compared to the control arm, oral dosing with F/TDF or F/TAF, significantly decreased p24 d15 in foreskin explant culture supernatants post-ex vivo challenge with HIV-1 BaL at both HVT and LVT [GMR (95% CI): 0.07 (0.05-0.11), p < 0.0001 and 0.07 (0.03-0.14), p < 0.0001 for HVT and LVT challenge, respectively; Fig. 2A Table S4). Calculation of the percentage reduction in p24 among participants receiving PrEP compared to those in the control arm demonstrates that nearly all had >75% reduction in p24 d15 (Supplementary Fig. S2A and C). Additional ex vivo dosing 20 h post-exposure to virus did not significantly increase the level of inhibition reached with oral dosing (Supplementary Fig. S2A and C; Supplementary Table S5). Furthermore, analysis of the viral replication curves suggested no evidence that additional ex vivo dosing further reduced p24 concentration in foreskin tissue observed with oral dosing (Supplementary Table S5).
In ex vivo challenged PBMCs, all oral PrEP arms significantly reduced p24 concentration compared to the control arm, with no evidence of differences between drug type, dosing or timing interval (Fig. 2B,D 20 In our study, [TFV-DP] >16 fmol/ 10 6 cells were obtained in 78% participants (72% 2 tablets; 84% 2 + 1) receiving on-demand F/TAF; whereas only 16% of participants receiving F/TDF achieved this threshold with a double dose, and 13% after a 2 + 1 dose. [FTC-TP] in PBMCs were in the range of previous studies 21 and there was no evidence that they were influenced by the concomitant TFV pro-drug.
Phosphorylated metabolites, TFV-DP and FTC-TP, were quantifiable in >90% of foreskin samples over 21 h post-dose (Fig. 3A)  Articles following LVT challenge (0.46 and 0.12, respectively). We examined correlations between drug concentrations and inhibitory activity. Presence of drug was associated with decreased p24 concentrations in the treated arms compared to the control arm; however, among participants receiving PrEP, there was no evidence of correlation between p24 and drug concentrations in any compartment.
To assess the drug distribution within CD4+ HIVtarget cells in foreskin, we conducted a case study with 28 foreskins from participants in the 2 + 1 dosing arms (Supplementary Table S8; Fig. 4A) (p-values determined by likelihood ratio tests). TFV-DP was detected in a total of 54% of CD4± samples analysed (36% CD4+; 71% CD4-). For those with detectable [TFV-DP] in CD4± fractions were similar between the TDF and TAF-based regimens, which reflected a lack of difference in [TFV-DP] seen in total foreskin tissue for this sub-set of individuals (p = 0.82; Fig. 4A). There was no evidence of a difference in the amount of TFV-DP/10 6 CD4 cells in isolated CD4+ and CD4-fractions (CD4+ = 24.  Table S9).
Evaluation of TFV-FTC and TAF-FTC PEP activity in foreskin was assessed in additional explants cut from control arm foreskins. A significant reduction in p24 concentrations was measured in cultures dosed 1, 24, 48 or 72 h post-ex vivo challenge with HVT and LVT titres (all p ≤ 0.001; Fig. 5 Positive 0 (0%) 1 (6%) 0 (0%) 0 (0%) 1 (6%) 0 (0%) 2 (13%) 2 (13%) 0 (0%) No  13  12  12  12  13  16  12  11  9 Yes 3 (19%) 3 (20%) 4 (25%) 4 (25%) 3 (19%) 0 (0%) 4 (25%) 5 (31%) 7 (44%) a 1 participant preferred not to say. b 2 participants preferred not to say.    The aim of the study was to provide guidance for dosing requirements and time to protection following starting PrEP. Our primary outcome demonstrated that an on-demand double dose regimen of F/TDF or F/ TAF, given on a single occasion, is protective between 5 and 21 h prior to insertive intercourse, and that post- coital dosing conferred additional protection in PBMCs but not tissue. Our data did not show clear superiority of TAF to TDF in protecting foreskins against HIV infections, however the higher TFV-DP intracellular concentrations we report in those who received TAF suggest that protection may be of longer duration, or that the dosing requirements for on-demand F/TAF are somewhat lower than for F/TDF. The results of this study align with findings from the social science arm of CHAPS, which clearly showed that young men in Uganda, Zimbabwe and South Africa favoured ondemand PrEP and if possible, a pre-coital only PrEP regime. 10 Both F/TDF and F/TAF were well tolerated and highly effective against ex vivo challenge of foreskin tissue and PBMCs with HIV-1 BaL . The added value in protection provided by post-exposure challenge in PBMCs but not in foreskin tissue is highly relevant in the discussion of whether pre-coital dosing is sufficient for insertive sex. The higher p24 concentrations observed in PBMCs compared to those in foreskin explants reflects the greater susceptibility of the systemic compartment to HIV-1 but the similar protection observed between the compartments is reassuring. As similar protection was observed at both time points, we were unable to ascertain whether protection occurs first in PBMCs or foreskin tissue. The range of p24 concentrations we report in foreskin tissue fell between those previously reported in rectal and vaginal explant cultures. 22 Our ex vivo challenge data suggest that the 2 + 1 PrEP regimen confers similar protection from HIV-1 infection as double dose PrEP and that the protection is sustained to at least 21 h after ingestion of the last PrEP dose. Our data are reassuring for young men who may not be able to predict the timing of sexual intercourse but would rather take a double dose of PrEP up to 21 h before having insertive sex.

Heard of PrEP for HIV prevention
PBMC metabolite exposures achieved in the F/TDF arms were comparable to those reported in the IPER-GAY single dose pharmacokinetic study. 21 By contrast, due to its preferential PBMC loading, 2 and 2 + 1 doses of F/TAF delivered high levels of TFV-DP that were both protective (>16 fmol/10 6 cells) and similar to TFV-DP exposures achieved with three (33%) and five (67%) daily doses per week of F/TDF. 23 [TFV-DP] and [FTC-TP] in foreskin were, in relation to other anatomical sites of HIV transmission, comparable to exposures previously reported in cervical and vaginal tissues. 24,25 Increased TFV-DP loading in PBMCs with F/TAF is well established, 25 and was replicated in this study. Interestingly, F/TAF also achieved higher [TFV-DP] in foreskin tissue than F/TDF; a finding that differs from the low TFV-DP detection rate previously seen in cervicovaginal tissues in participants treated with a single (25 mg) dose of TAF. 24,25 This suggests that distribution of TAF and its conversion to TFV-DP in foreskin cannot be inferred from the vaginal compartment, reenforcing the need to evaluate drug kinetics within distinct anatomical sites and patient populations of interest.
Intracellular TFV-DP accumulated in foreskin, which is potentially attributed to its long intracellular half-life in mucosal tissues (34-53 h in cervicovaginal tissues 26 ). By contrast, FTC-TP exhibited a more rapid distribution in foreskin tissue but declined more rapidly. FTC-TP has previously been shown to decline in cervicovaginal tissues at a faster rate than TFV-DP. 27 There was an added pharmacokinetic benefit of giving an extra dose the following day as participants receiving a 2 + 1 regimen had higher [TFV-DP] in foreskin, although mucosal FTC-TP levels were unchanged. Cottrell and colleagues found no evidence of dose proportionality, for either parent drug or metabolite, in the female genital tract and in colorectal tissues after giving Articles escalating single doses of TDF and FTC; and in PBMC, TFV-DP concentrations increased proportionally but FTC-TP did not. 28 This study has successfully measured phosphorylated antiretroviral nucleosides in CD4± cells isolated from foreskin tissue. Data from this small sub-study revealed that, unlike the PBMCs compartment, dosing with TAF did not confer preferential distribution of TFV-DP into foreskin HIV target cells. The lack of correlation between PBMCs and foreskin CD4+ fractions, further supports the notion that CD4+ cells are a distinct cellular sub-population. However, the small sample size, low detection rate, and high inter-subject variation mean that these preliminary data should be interpreted with caution.
A key strength of the trial design is the ability to assess several important PrEP variables including PrEP drug, PrEP dose and timing, and link tissue and cellular pharmacokinetics to pharmacodynamics. Unlike studies with HIV incidence as an outcome, no participants were exposed to additional risk of HIV due to group allocation.
Our study had some limitations. Firstly, this was an open-label study with blinding of arm allocation to laboratory staff but not participants or clinical personnel. Second, one foreskin specimen and two PBMC specimens from the control arm were not infected following ex vivo challenge with LVT, which affected the baseline control of infection; however, this reflects the donor-todonor variability of in vivo susceptibility to HIV transmission. Third, HVTs routinely used for ex vivo challenge of foreskin explants, could not be used in PBMCs. Fourth, our sample size was relatively small, therefore we cannot rule out the possibility that there were differences between the PrEP trial arms that we were not powered to detect. Fifth, the study was not designed as a drug titration, prohibiting a more in-depth PK-PD correlation analysis among treated arms. The lack of PK-PD correlation could be due to activity of FTC which could be masking differences between TFV and TAF. The tissue explant model cannot appropriately metabolize the formulated version of TFV for oral dosing, hence, ex vivo dosing is performed with the base compound. Furthermore, tissue explants have limited capacity to demonstrate sterilizing protection, and progressively lose their architecture; however, CD4:CD8 T cell ratios and sufficient viability are maintained during culture to sustain viral replication. 29 Finally, the location of the VMMC clinics in relation to the central processing laboratories meant it was not feasible to immediately section and "snap freeze" foreskin tissue at time of collection; instead, the entire foreskin was transported in media to the laboratory. This potentially accounts for the large proportion of tissue samples that had undetectable drug (81% TFV; 38% FTC), having previously demonstrated that drug loss from a foreskin tissue explant during culture in media is extensive. 18 In an era when efficacy studies for HIV protection are large and prohibitively expensive, evidence from phase II studies of this nature will maximise the chance of identifying the most efficacious dosing strategy and filling data gaps around onset and offset of protection. This factorial design using the p24 protein as a surrogate endpoint can be used to efficiently select drug combinations and dosing regimens for testing in phase III, while obviating the unnecessary costs of futile regimens. The results from this study and future ex vivo HIV-challenge trials could inform future trials drug, dose and schedules that could offer the best protection against HIV.

Contributors
CH and JS contributed equally to conceptualisation, formal analysis, investigation, methodology, resources, supervision, validation, writing original draft; LE: conceptualisation, formal analysis, investigation, methodology, resources, supervision, validation, writingoriginal draft; LL: project administration, resources, supervision; DO, PS and RM: resources; ASS: project administration, resources; ADP, PN, TBS, GO, SM, AA, SDP and SP: investigation; BA: project administration, resources; KOH and CC: methodology; JS and HW: conceptualisation; SK: conceptualisation, supervision, validation, writingoriginal draft; FC: conceptualisation, resources, supervision, validation, writingoriginal draft; CMG: conceptualisation, resources, supervision; PK: conceptualisation, supervision; ELW: data curation, formal analysis, supervision, validation, writingoriginal draft; NM and JF contributed equally to conceptualisation, funding acquisition, resources, supervision, writingoriginal draft. All authors contributed to the writing-review & editing. All authors read and approved the final version of the manuscript. Participants enrolled in the CHAPS cohort provided their time and consented for the donation of resected foreskin to the study.D ata sharing statement Deidentified participant data and a corresponding data dictionary will be available together with the study protocol, with publication and upon request to the corresponding author. This will be made available on LSHTM Data Compass repository. The Trial Management Group will approve data sharing requests.